PROJECT SUMMARY/ABSTRACT Cardiovascular diseases (CVD) are the leading cause of death in the US and worldwide. In 2009 alone, more than 80 million Americans suffered from CVD with associated cost $313 billion. The development of CVD and other chronic circulatory disorders has been associated with exposure to environmental toxicants. The toxicants can alter bioavailability of a key regulator of vascular function ? nitric oxide (NO), which is produced by endothelial cells. The impairment of vascular endothelial NO production can contribute to the development of chronic circulatory disorders. However, this does not explain why some individuals develop the disease and others do not. Accordingly, the National Institute of Environmental Health Sciences leads the efforts to understand the contribution of individual susceptibility to complex diseases, such as CVD. To aid these efforts, Creative Scientist, Inc. (CSI) is developing advanced tools enabling the generation of data on individual vascular susceptibility to environmental toxicants. CSI employs isolated Endothelial Colony-Forming Cells (ECFC), which are unique primary cells involved in both embryonic and adult vasculogenesis. ECFCs can be isolated from blood and cryopreserved. Donor-specific nature and robust growth potential of ECFCs allow for examining tens of thousands of chemicals in cells derived from any number of people. CSI has developed proprietary protocols and cell growth media supplement to isolate ECFCs from small volumes of blood. Our goal is to commercialize an NO testing service using novel and highly specific NO reagent and a high content imaging technique. In Aim 1, we will determine sensitivity and selectivity of our novel NO-specific fluorescent probe. Completion of this Aim will demonstrate the utility of our reagent for NO measurement in ECFCs. In Aim 2, we will determine reproducibility of NO measurement in different ECFC lineages throughout the cell culture. Completion of Aim 2 will establish the utility of ECFC for an identification of NO inhibitors. In Phase II, we will test assay?s capability to detect variability of responses to 1,000 toxicants in a collection of ECFCs derived from up to 500 donors. Our ultimate goal is provide a commercial service to regulatory bodies and pharmaceutical companies aiming to evaluate potential vascular toxicity of environmental toxicants and drug candidates in a population and, in particular, trying to identify both sensitive and resistant individuals and/or population sub-groups.